US11499156B2 - Biomolecule imaging method using aptamer - Google Patents

Biomolecule imaging method using aptamer Download PDF

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US11499156B2
US11499156B2 US16/608,645 US201816608645A US11499156B2 US 11499156 B2 US11499156 B2 US 11499156B2 US 201816608645 A US201816608645 A US 201816608645A US 11499156 B2 US11499156 B2 US 11499156B2
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aptamer
odn
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US20200157543A1 (en
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Jung Hwan Lee
Jong In Kim
Jong Hun Im
Jong Ook LEE
Jin Woo Kim
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Interoligo Corp
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    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
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    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0404—Lipids, e.g. triglycerides; Polycationic carriers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the present invention relates to a biomolecule imaging method using aptamer, and more particularly, to a method for obtaining images by using aptamer labeled with isotope and binding the same to a human epidermal growth factor receptor 2 (HER2) expressing cell line.
  • HER2 human epidermal growth factor receptor 2
  • the etymology of aptamer was derived from the Latin meaning of “aptus (exactly right)” and the Greek meaning “meros (partial).”
  • the aptamer is a single-stranded nucleic acid having a DNA sequence consisting of about 20 to 90 bases.
  • aptamer highly specific to a target molecule and having a high affinity thereto is screened through an artificial evolution method such as in vitro SELEX (Systematic Evolution of Ligands by Exponential Enrichment) as an aptamer excavation technology. Therefore, the aptamer is regarded as a very suitable reagent to determine or find a degree of expression of specific molecules to be targeted by the aptamer.
  • the aptamer has advantages as compared to the antibody.
  • the aptamer is a reagent relatively newly developed in diagnosis fields.
  • a number of aptamers for a wide variety of targets including thrombin, nucleolin, PSMA, TNC and virus origin proteins have been developed.
  • VEFG target aptamer was developed and approved as elderly macular degeneration therapeutic agents by the FDA in 2004.
  • Recently, so many types of aptamers are under development in pre-clinical and clinical phases and a number of experiments relevant to diagnosis and treatment are in the process.
  • HER2 is a cancer gene very well known in the art, which is increased or over-expressed in about 15 to 30% of breast cancer. Further, this is a factor associated with high recurrence and poor prognosis of different cancers.
  • transtuzumab and pertuzummab for targeting HER2 currently exist as therapeutic monoclonal antibodies well known and available in the art, and have been found to be effective in clinical applications.
  • HER2 targeting DNA/RNA aptamers were disclosed through traditional SELEX methods and cell-based SELEX. Moreover, examples of pharmaceutically utilizing cancer inhibitory properties of the HER2 aptamers have been recently reported.
  • molecule images may be a non-invasive method that enables real-time visualization of biochemical events in a cellular molecular level in regard to living cells or tissues or objects without damage.
  • the aptamer modified into a magnetic nano-material or fluorescent material may be provided as a preferred substance for targeted fluorescence imaging or magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • Some in vivo MRI studies demonstrated efficiently targeted cancer in mice having cancer.
  • PET is distinctly more advantageous in a diagnostic aspect than anatomical techniques such as computed tomography (CT) and MRI. In clinical applications, PET is broadly used in basic research and preclinical fields.
  • the PET may be used to verify or validate analysis of new radio-therapeutics, therapeutic efficacy of novel therapeutic agents and in vivo distribution of drugs.
  • Merits of PET may include probe depth, superior sensitivity, quantitative data and convertibleness (i.e., phase progress) from pre-clinical trials to clinical trials. That is, the PET is a representative molecule imaging device that can detect biochemical changes in a target level of living biomolecules and is highly sensitive, thereby being used in a wide range of applications including basic science and pre-clinical area.
  • Cancer targeting using aptamer is a biomolecule imaging technique proposed in recent years, and for example, many researchers including Hicke et. al. have adopted aptamer in molecule imaging.
  • Aptamer is one of nucleic acids and a material with high specificity and affinity to a target molecule. It is an object of the present invention to provide molecular images in vivo using radioactive isotope or fluorescent dye-labeled aptamer.
  • FIG. 1 is a mechanism schematic view illustrating radioactive isotope or fluorescence-labeled ERBB2 aptamer.
  • HER2 aptamer labeled with a radioactive isotope or fluorescent dye is used for in vivo imaging.
  • ERBB2 aptamer In flow cytometric analysis, ERBB2 aptamer is almost not bound to MDA-MB231 cell line without expression of HER2, but may have very high affinity to BT474 as a HER2 expressing cell line. Similarly, it is observed from images obtained by a confocal microscope that the aptamer is bound to HER2 expressed breast cancer cell line, while showing only minimum binding to HER2 non-expressing cells. Molecular images of positron emission tomography for a mouse transplanted in vivo with BT474 cancer cell line have demonstrated a significant increase in intake of 18 F-labeled HER2-specific ERBB2 aptamer. ERBB2 aptamer may be preferentially bound to HER2 expressed breast cancer cell line both in vitro and in vivo, and the reason is that HER2 structure is possibly recognized on the surface of the cells.
  • ERBB2 aptamer labeled with a radioactive isotope such as 18 F or a fluorescent dye may recognize HER2 expression in human breast cancer cells and enable adequate visualization.
  • FIG. 1 is a schematic diagram illustrating a mechanism of radiation or fluorescence-labeled ERBB2 aptamer.
  • FIG. 3 illustrates results of identifying the complementary base pairing between cholesteryl-[AP001-24]-ODN-idT or cholesteryl-[AP001-24]-ODN aptamer and fluorescence-labeled cODN (cODN-Cy5), using 3% agarose gel at 50, 55 and 60° C. (black: aptamer, and red: cODN-Cy5).
  • FIG. 4 illustrates results of identifying the complementary base pairing between cholesteryl-[AP001-24]-ODN-idT, cholesteryl-[AP001-24]-ODN, PEGylated-[AP001-25]-ODN-idT or PEGylated-[AP001-25]-ODN aptamer and fluorescence-labeled cODN (cODN-Cy5), which were heated to 95° C., using agarose gel.
  • FIG. 5 illustrates confocal image results of KPL4, N87 and SK-BR cell lines treated with R-[ERBB2 aptamer]-X-hy(bp)-Cy5, including confocal microscopic images of [AP001-24]-hy(bp)-Cy5 and [AP001-25]-hy(bp)-Cy5 aptamer in HER2-positive cell line, in particular: (a) treatment of KPL4, HER2-positive breast cancer cell line with Cy-labeled aptamer; (b) treatment using the same aptamer in N87 cancer cell line; and (c) treatment using the same aptamer in SK-BR-3 cancer cell line (marker DAPI: blue, and Cy5-aptamer: red).
  • FIGS. 6A and 6B illustrate results of FACS analysis of KPL4, N87 and SK-BR cell lines treated with R-[ERBB2 aptamer]-X-hy(bp)-Cy5, respectively.
  • FIGS. 7A and 7B illustrate results of microPET images of [AP001-24]-hy(bp)-L-F 18 .
  • FIGS. 8A and 8B illustrate results of microPET images of [AP001-24]-idT-hy(bp)-L-F 18 .
  • FIGS. 9A and 9B illustrate results of microPET images of cholesteryl-[AP001-24]-hy(bp)-L-F 18 .
  • FIGS. 10A and 10B illustrate results of microPET images of cholesteryl-[AP001-24]-idT-hy(bp)-L-F 18 .
  • FIG. 11 illustrates results of microPET images of PEGylated-[AP001-24]-hy(bp)-L-F 18 .
  • FIG. 12 illustrates results of microPET images of PEGylated-[AP001-24]-idT-hy(bp)-L-F 18 .
  • FIG. 13 is comparative images of [AP001-24]-hy(bp)-L-F 18 , [AP001-24]-idT-hy(bp)-L-F 18 , cholesteryl-[AP001-24]-hy(bp)-L-F 18 and cholesteryl-[AP001-24]-idT-hy(bp)-L-F 18 in mice having KPL4 cancer.
  • FIG. 14 illustrates determination of a degree of HER2 expression in human breast cancer cell line by Western blot.
  • FIGS. 15A and 15B illustrate flow cytometric analysis of breast cancer cell line using HER2 antibody and ERBB2 aptamer (AP001-25), wherein: (a) a viscosity table shows fluorescent signals from antibodies to BT474 (HER2-positive cell line) and MDA-MB231 (HER2-negative cell line), with ERBB2 aptamer (AP001-25) (red) or DNA sequence of a control group (blue); and (b) flow cytometric analysis graphs of both cell lines using the antibody, ERBB2 aptamer (AP001-25) and a negative control are included.
  • a viscosity table shows fluorescent signals from antibodies to BT474 (HER2-positive cell line) and MDA-MB231 (HER2-negative cell line), with ERBB2 aptamer (AP001-25) (red) or DNA sequence of a control group (blue); and (b) flow cytometric analysis graphs of both cell lines using the antibody, ERBB2 aptamer (AP001-25) and
  • FIGS. 16A and 16B illustrate confocal microscopic images of the selected ERBB2 aptamer (AP001-25) in HER2-positive cell line, in particular: (a) treatment of BT474, HER2 positive breast cancer cell line with FITC marker aptamer; and (b) treatment using the same ERBB2 aptamer (AP001-25) in MDA-MB231 cancer cell line (marker DAPL: blue, and FITC-ERBB2 aptamer: green).
  • FIG. 17 illustrates in vivo PET images representing 18 F-labeled ERBB2 aptamer ⁇ [AP001-25]-hy(bp)-L-F 18 ⁇ in mice having BT474 cancer, wherein Hy(bp) indicates ODN/cODN hybridization as the hybridization (base pairing).
  • FIGS. 19A to 19C illustrate in vivo PET images representing 18 F-labeled ERBB2 aptamer ⁇ [AP001-25]-hy(bp)-L-F 18 ⁇ in mice having HER2-positive and negative cancers, respectively, in particular: (a) HER2 over-expressing BT474 cancer (left armpit); (b) HER2 negative MDA-MB231 cancer (right armpit) wherein (a) shows more increased intake than in (b); and (c) count per minute (CPM) and injection amount per gram (% ID/g) into tumor tissue are calculated with 18 F-labeled ERBB2 aptamer.
  • CPM count per minute
  • % ID/g injection amount per gram
  • FIG. 20 illustrates H&E and IHC staining of HER2 (original magnification 400 ⁇ )
  • ERBB2 aptamer specifically bound to HER2 receptor in relevant to breast cancers which is used in the present invention, has a DNA sequence of 5′-TCAGCCGCCAGCCAGTTC-[core sequence]-GACCAGAGCACCACAGAG-3′ wherein the number ‘6’ in the core sequence or ‘n’ in the attached DNA sequence listing represents NaptyldU.
  • HER2 aptamer labeled with a radioactive isotope for example, 18 F, 32 P, 123 I, 89 Zr, 67 Ga, 201 Tl and 111 In-111, or a fluorescent dye, for example, a cyanine fluorescent dye such as Cy3, Cy5, Cy7, etc. was utilized for in vivo imaging.
  • radioactive isotope for example, 18 F, 32 P, 123 I, 89 Zr, 67 Ga, 201 Tl and 111 In-111
  • a fluorescent dye for example, a cyanine fluorescent dye such as Cy3, Cy5, Cy7, etc. was utilized for in vivo imaging.
  • evaluation of target specificity for in vivo molecule imaging and potential clinical application have been performed using ERBB2 aptamer labeled with the radioisotope or fluorescent dye.
  • ERBB2 aptamer for a human epidermal growth factor receptor 2 was labeled with 18 F-fluoride isotope.
  • the aptamer was compared with a control aptamer by flow cytometry and confocal microscope.
  • the 18 F-labeled HER2-specific ERBB2 aptamer was subjected to positron tomography thus to obtain biomolecular images of the mice transplanted with BT474 or KPL4 cells over time.
  • HER2 expressed human breast cancer cell lines e.g., BT474, KPL4, N89 and SK-BR-3 were used for in vitro and in vivo experiments. Further, a human breast cancer cell line MDA-MB231 was used as a control group. All cell lines were purchased from ATCC and incubated and maintained in MEM medium containing 10% FBS.
  • a cell lysate including a protease inhibitor was incubated on ice for 30 minutes.
  • the resulting cell lysate was purified by centrifugation at 4° C. for 20 minutes.
  • the cell lysate was quantified by Bradford method, followed by separation of 30 ⁇ g protein extract from the respective samples through electrophoresis using 10% SDS-PAGE. Then, the resulting product was transferred to a nitrocellulose membrane and subjected to photosensitization on x-ray film with ECL, using HER2 antibody and the control group, that is, a beta-action antibody as a probe.
  • the RBB2 aptamers in particular, AP001-24 has a binding affinity (Kd) of 3.1 nM to a target and AP001-25 has a binding affinity of 0.9 nM.
  • aptamer hybridization For aptamer hybridization, synthesis including a fully matching sequence, that is, ODN (5′-CAGCCACACCACCAG-3′) (SEQ ID NO: 36) at 3′ in each of the ERBB2 aptamers ⁇ [AP001-24] and [AP001-25] ⁇ was performed.
  • Aptamer synthesis was performed by a solid phase synthesis process through phosphoramidite coupling reaction, and after the synthesis, the product was reacted in a t-butylamine:methanol:water (1:1:2 v/v/v) solution at 70° C. for 5 hours, thus to obtain a complete aptamer through cleavage and deprotection processes, followed by drying the same.
  • the synthesized aptamer was isolated by HPLC [C18 column (Waters, Xbridge OST C18 10 ⁇ 50 mm, 260 nm] and then was subjected to measurement of a molecular weight by means of ESI MS mass spectrometer (Qtrap2000, ABI).
  • 11 th aptamer in Table 1 corresponds to AP001-24.
  • each of aptamers that is, CAG-3′ ⁇ each of aptamers (SEQ ID NOs: 1-35) in Table 1-ODN ⁇ was synthesized by the same procedures as described in the above section for ⁇ [AP001-24]-ODN ⁇ synthesis.
  • the Cy5-labeled ERBB2 aptamer that is, ⁇ R-[ERBB2 aptamer]-X-hy(bp)-Cy5 ⁇ was prepared in the following manner.
  • cODN-Cy5 and [ERBB2 aptamer]-ODN in equal moles were put in an annealing buffer (PBS).
  • PBS annealing buffer
  • a concentration of MgCl 2 was controlled to reach a final concentration of 10 mM.
  • This reaction product was left at 95° C. for 5 minutes, and then slowly cooled at room temperature.
  • Hybridization efficiency of cODN-Cy5 and [ERBB2 aptamer]-ODN was assessed by electrophoresis (Typhoon FLA 7000 3% agarose gel analysis) and HPLC (XBridge OST analytical column (2.5 ⁇ m, 4.6 ⁇ 50 mm, Waters, 254 nm, 0.1M TEAA/acetonitrile).
  • each of these aptamers was mixed with cODN-Cy5 in 1:1 ratio and heated at 95° C. for 5 minutes to bind together, followed by assessment of the binding in the same manner as described above.
  • Synthesis of 18 F-labeled cODN was performed on the basis of the process already reported in the art (see reference 24). After generating no-carrier-added 18F-fluoride ions in a synthesis device (Tracerlab FXFN, GE Healthcare, Milwaukee, Wis., USA) and reacting the same with mesylate (at 100° C. for 10 minutes), 18 F-fluoro-PEG-azide (18F-FPA) was purified by using HPLC.
  • the synthesized 18 F-labeled cODN (cODN-L-F 18 ) was purified by using HPLC H (Xbridge OST C18 10 ⁇ 50 mm, an eluent of acetonitrile/0.1M TEAA in 5:95 to 95:5 over 20 minutes, flow rate: 5 mL/min, and UV (254 nm)).
  • cODN-L-F 18 and [ERBB2 aptamer]-ODN in equal moles were put in an annealing buffer (PBS).
  • PBS annealing buffer
  • a concentration of MgCl 2 was controlled to reach a final concentration of 10 mM.
  • This reaction product was left at 95° C. for 5 minutes, and then slowly cooled at room temperature.
  • Hybridization efficiency of cODN-L-F 18 and [ERBB2 aptamer]-ODN was assessed by using HPLC (XBridge OST analytical column (2.5 ⁇ m, 4.6 ⁇ 50 mm, Waters, 254 nm, 0.1M TEAA/acetonitrile). These products were combined at a hybridization rate of 98% or more.
  • BT474, KPL4, N87, SK-BR-3 and MDA-MB231 cell lines were dispensed on a coverslip and incubated overnight.
  • the grown cells were carefully washed and incubated by treatment using fluorescence-labeled ERBB2 aptamer ⁇ R-[ERBB2 aptamer]-hy(bp)-Cy5 ⁇ at a concentration of 250 mM.
  • the product was carefully washed, followed by loading a culture medium containing DAPI on a slide. Then, florescence thereof was observed by an LSM 700 confocal microscope.
  • Microscope setting was performed as follows: a 488 laser was used for FITC observation; excitation and emission were observed using BP490-555; a 639 laser was used for Texas red; and emission was observed using an LP640 filter.
  • ERBB2 over-expressing breast cancer cell lines e.g., KPL4, N87 and SK-BR-3 were dispensed on a coverslip and incubated overnight.
  • the grown cells were carefully washed and incubated by treatment using a sample prepared of Cy5 fluorescence-labeled ODN bound to ERBB2 aptamer using complementary base pairing.
  • the product was carefully washed, followed by loading a culture medium containing DAPI on a slide. Then, florescence was observed by an LSM 700 confocal microscope.
  • ERBB2 aptamer Specificity of ERBB2 aptamer was verified by a fluorescence activated cell separation method using a flow cytometry system (BD Biosciences). Appropriate numbers of BT474, KPL4, N87, SK-BR-3 or MDA-MB231 cancer cell lines were sub-cultured on a Petri-dish to grow the same to about 80%. The grown cells were treated with trypsin and washed with PBS, followed by binding fluorescence-labeled ODN to ERBB2 aptamer through a complementary base generated according to a temperature. The cells were treated with the binding-completed sample.
  • Both of the ERBB2 aptamer ⁇ R-[ERBB2 aptamer]-hy(bp)-Cy5 ⁇ and the control group, that is, 1% FES containing antibody were treated at 4° C. for 30 minutes, respectively.
  • the completely treated sample was washed, followed by measurement and analysis of the bound ERBB2 aptamer by a fluorescence activated cell separation method.
  • 17 ⁇ -estradiol pellets were subcutaneously implanted into a side region of the neck of a 4 week-old Balb/c nude mouse so that estrogen is released in a sufficient amount to potentially induce a cancer.
  • BT474 or KPL4 human breast cancer cell line was subcutaneously implanted in 7 ⁇ 10 6 cells per mouse. After allowing the cancer to develop for 3 weeks, cancer growth was measured using a caliper.
  • KPL4 cells as the human breast cancer cell line were subcutaneously implanted in 1 ⁇ 10 5 cells per mouse. Thereafter, occurrence of cancer was induced.
  • F 18 radioisotope-labeled ERBB2 aptamer was injected to a mouse, and after 60 minutes, static images were obtained by Inveron microPET scanner (Siemens, Knoxville, Tenn., USA) for 10 minutes.
  • Inveron microPET scanner Siemens, Knoxville, Tenn., USA
  • the mouse was breathing anesthetized with 2% Isoflurane, followed by 7.4 MBq of F 18 radioisotope-labeled ERBB2 aptamer injection into a tail vein.
  • the obtained listmode data is converted into synogram and re-configured by 3D Ordered Subset Expectation Maximization (OSEM) algorithm, followed by assessment using ASIpro (Concord Microsystems Inc., Knoxville, Tenn.).
  • PET After intravenous injection of F 18 radioisotope-labeled ERBB2 aptamer to a mouse having tumor grown by injection of human breast tumor cells, PET was executed using inveon PET of Siemens (Knoxville, Tenn.). The injected amount was 13.7 ⁇ 1.1 MBq (370 ⁇ 30 uCi), and dynamic PET study was implemented for 30 minutes according to ten 1-minute image and four 5-minute image protocols. These two stationary studies were conducted for 10, 60, 90 and 120 minutes, respectively, after the injection. Partial quantification of PET signals was executed by AMIDE software. Images were practically gained by false-color-scale in proportional to the tissue concentration (% ID/g) of a positron labeling probe. Red represents the highest concentration, while yellow, green and blue correspond to gradually lower concentrations.
  • PET images are shown in FIGS. 7A to 13 .
  • HER2 antibody is very specifically bound to HER2-positive BT474 cell line using the flow cytometry.
  • ERBB2 aptamer is very weak in MDA-MB231 cell line, whereas in BT474 cell line it is strongly bound thereto.
  • binding of the aptamer to any cell line could not be seen in random oligonucleotides.
  • FIGS. 16A and 16B Binding of ERBB2 aptamer to cells was further assessed by a confocal microscope ( FIGS. 16A and 16B ).
  • BT474 HER2-positive breast cancer cell line was treated with the aptamer. Since ERBB2 aptamer was fluorescence-labeled, fluorescence was observed on the surface of the cell, and HER2 structure present on the cell surface was identified. Fluorescence exhibited by the aptamer was observed along a cellular membrane, while MDA-MB231 cell line as a negative control group did not indicate any fluorescent signal and thus was determined not to include HER2. Accordingly, it was observed that ERBB2 aptamer could be bound to HER2-positive breast cancer cell line, but is minimally bound to HER2-negative cells.
  • the intake of 18 F-labeled ERBB2 aptamer in the cancer was 0.62 ⁇ 0.04 per hour.
  • Study on in vivo distribution demonstrated that the kidney and the intestine are two major discharge routes of 18 F-labeled ERBB2 aptamer.
  • FIGS. 19A to 19C illustrate images of 18 F-labeled ERBb2 aptamer in mice having HER2-positive and negative cancers, respectively.
  • HER2 over-expressing BT474 cancer shows higher isotopic intake than HER2-negative MSA-MB231 cancer by comparison.
  • total activity (nCi) among VOIs (voxels- or volumes-of-interest) was calculated.
  • T/M tumor/muscle
  • BT474 cancers excised from separate mouse groups showed high HER2 expression, whereas MDA-MB231 cells have lower HER2 expression ( FIG. 20 ). It was observed that BT474 cancer cells (upper row) show higher staining for HER2 in a cellular membrane than MDA-MB231 cell line (lower row) by comparison.
  • HER2 targeting ERBB2 aptamer was successfully PET-imaged in vivo.
  • the present invention is the first case to execute HER2 target PET imaging using ERBB2-specific aptamer.
  • PET images demonstrated that ERBB2 aptamer may recognize HER2 in vivo and relatively distinctively show the cancer. Based on these results, the radio-labeled ERBB2 aptamer may be applied to targeted treatment of HER2-positive breast cancer cell line or potentially applied to determination of appropriate therapeutic methods for the same.

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090004667A1 (en) 2007-01-16 2009-01-01 Somalogic, Inc. Method for generating aptamers with improved off-rates
KR20110086433A (ko) 2010-01-22 2011-07-28 성균관대학교산학협력단 Ｈｅｒ－２ 과발현 유방암 세포 또는 조직에 특이적으로 결합할 수 있는 핵산 압타머 및 그 용도
KR20130003071A (ko) 2011-06-24 2013-01-09 포항공과대학교 산학협력단 Ｅｒｂｂ2 수용체에 선택적으로 결합하는 압타머 및 이의 용도
KR20130012140A (ko) 2013-01-09 2013-02-01 대한민국 (식품의약품안전청장) Ｅｒｂｂ2 수용체에 선택적으로 결합하는 압타머 및 이의 용도
KR101405440B1 (ko) 2012-07-31 2014-06-20 연세대학교 산학협력단 인테그린αｖβ３에 특이적 압타머 및 이의 용도
KR20150045592A (ko) 2013-10-21 2015-04-29 연세대학교 산학협력단 다양한 압타머에 적용 가능한 방사성동위원소 표지된 핵산 구조체 개발 및 표지방법

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090004667A1 (en) 2007-01-16 2009-01-01 Somalogic, Inc. Method for generating aptamers with improved off-rates
KR20110086433A (ko) 2010-01-22 2011-07-28 성균관대학교산학협력단 Ｈｅｒ－２ 과발현 유방암 세포 또는 조직에 특이적으로 결합할 수 있는 핵산 압타머 및 그 용도
KR20130003071A (ko) 2011-06-24 2013-01-09 포항공과대학교 산학협력단 Ｅｒｂｂ2 수용체에 선택적으로 결합하는 압타머 및 이의 용도
US20150005368A1 (en) 2011-06-24 2015-01-01 Korea Food & Drug Administration Aptamer which selectively binds to erbb2 receptor and uses thereof
US9309515B2 (en) * 2011-06-24 2016-04-12 Postech Academy-Industry Foundation Aptamer which selectively binds to ERBB2 receptor and uses thereof
KR101405440B1 (ko) 2012-07-31 2014-06-20 연세대학교 산학협력단 인테그린αｖβ３에 특이적 압타머 및 이의 용도
KR20130012140A (ko) 2013-01-09 2013-02-01 대한민국 (식품의약품안전청장) Ｅｒｂｂ2 수용체에 선택적으로 결합하는 압타머 및 이의 용도
KR20150045592A (ko) 2013-10-21 2015-04-29 연세대학교 산학협력단 다양한 압타머에 적용 가능한 방사성동위원소 표지된 핵산 구조체 개발 및 표지방법

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
Brian J. Hicke et al., "Tumor Targeting by an Aptamer", The Journal of Nuclear Medicine, vol. 47, No. 4, pp. 668-678, 2006.
Dion A. Daniels et al., "A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment", Proceedings of the National Academy of Sciences of the USA, vol. 100, No. 26, pp. 15416-15421, 2003.
Guizhi Zhu et al., "Combinatorial Screening of DNA Aptamers for Molecular Imaging of HERZ in Cancer", Bioconjugate Chemistry, 28, pp. 1068-1075, 2017.
International Search Report for PCT/KR2018/004770 dated Aug. 29, 2018.
Kang, Hye Suk et al., "Isolation of RNA Aptamers Targeting HER-2-overexpressing Breast Cancer Cells Using Cell-SELEX", Bulletin of the Korean Chemical Society, vol. 30, No. 8, pp. 1827-1831, 2009.
Kim , Mee Young et al., "In Vitro Selection of RNA Aptamer and Specific Targeting of ErbB2 in Breast Cancer Cells", Nucleic Acid Therapeutics vol. 21, No. 3, pp. 173-178, 2011.
Moosavian S A et al., "Development of RNA Aptamers as Molecular Probes for HER2+ Breast Cancer Study Using Cell-SELEX", Iranian Journal of Basic Medical Sciences, vol. 18, pp. 576-586, 2015.
Notice of Allowance dated Dec. 1, 2019 from Korean Patent Office in a counterpart Korean Patent Application No. 10-2018-0046550 (all the cited references are listed in this IDS.) (English translation is submitted herewith).
Office action dated Dec. 15, 2020 from Japan Intellectual Property Office in a counterpart Japanese Patent Application No. 2019-557747(all the cited references are listed in this IDS.) (English translation is also submitted herewith.).
Orit Jacobson et al., "PET imaging of Tenascin-C with a Radiolabeled single-strand DNA aptamer", Journal of nuclear medicine: official publication, Society of Nuclear Medicine, 56: 616-21, 2015.
Park Jun Young et al. "Hybridization-based aptamer labeling using complementary oligonucleotide platform for PET and optical imaging", Biomaterials, Elsevier, Amsterdam, NL, vol. 100, May 24, 2016 (May 24, 2016), pp. 143-151, XP029570457 (English translation is submitted herewith.).
PARK JUN YOUNG; LEE TAE SUP; SONG IN HO; CHO YE LIM; CHAE JU RI; YUN MIJIN; KANG HYUNGU; LEE JUNG HWAN; LIM JONG HOON; CHO WON GIL: "Hybridization-based aptamer labeling using complementary oligonucleotide platform for PET and optical imaging", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, vol. 100, 24 May 2016 (2016-05-24), AMSTERDAM, NL , pages 143 - 151, XP029570457, ISSN: 0142-9612, DOI: 10.1016/j.biomaterials.2016.05.035
Shawn E. Lupoid et al., "Identification and Characterization of Nuclease-stabilized RNA Molecules That Bind Human Prostate Cancer Cells via the Prostate-specific Membrane Antigen", Cancer Research 62, pp. 4029-4033, 2002.
The extended European Search Report for EP18791814.9 dated Jan. 14, 2021 from European patent office in a counterpart European patent application.

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